The development of more effective vaccines requires an understanding of the requirements for strong short and long-term immune responses. Memory cell formation is a hallmark of the adaptive immune system and CD4+ T cells play a central role in the generation of productive recall responses and protective immunity. CD4+ T cells coordinate the adaptive and innate immune responses to infection, but their role as memory cells is not well understood. An unresolved question is what CD4+ TCR:pMHC affinity leads to optimal CD4+ memory cell formation. To address this, we have recently generated two CD4+ TCR transgenic mouse lines that are specific for the same naturally occurring epitope from Listeria monocytogenes. The TCRs from these mice lines (LLO118 and LLO56) differ by only 15 amino acids and while LLO118 and LLO56 T cells have a similar in vitro response to antigen, their in vivo responses to infection are strikingly different. LLO118 has a stronger primary response whereas LLO56 has a much stronger memory response. Understanding why these TCRs cause such different T cell responses provides a novel system to understand how to improve vaccines and immunotherapies. In Aim 1, we have generated four L. monocytogenes mutants with different capacities to stimulate LLO118 and LLO56 T cells to test the role of TCR:pMHC binding affinity in CD4+ primary and memory responses. Additionally, we unexpectedly found that LLO56, the T cell with the best memory response, has higher levels of pro-apoptotic staining (Annexin V) after antigen stimulation. In Aim 2 we want to confirm this using additional apoptotic markers and determine how cell death is related to memory cell formation. Finally, we have found expression differences in two T cell co-receptors (CD5 and CD6) that negatively regulate T cell activation. These are both up-regulated in LLO56, which has the weaker primary response but stronger memory response, and in Aim 3 we will be using CD5 knockout mice and CD6L (CD166) knockout mice to determine their role in controlling the primary and memory responses of LLO118 and LLO56. Our studies have the potential to identify the characteristics of CD4+ T cells responsible for differential immune responses and this could be highly relevant for improving therapeutic and protective vaccine design and immunotherapies.